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NOTE
A synthesis of tissue-preservation effects on
carbon and nitrogen stable isotope signatures
Helen C. Sarakinos, Michael L. Johnson, and M. Jake Vander Zanden
Abstract: Stable-isotope analysis is a powerful method for characterizing flows of mass and energy through ecosystems. Long-term food-web studies using stable isotopes are valuable but rare because the required samples are not
readily available. We examine the feasibility of using preserved specimens from natural-history collections as a source
of long-term data for food-web studies and test whether chemical preservation affects the stable-isotope signature of
tissues. We experimentally determined the effects of tissue preservation and fixation with 75% ethanol and 10% formalin, respectively, on δ13C and δ15N of three aquatic consumers: Sacramento sucker, Catostomus occidentalis, Asian
clam, Corbicula fluminea, and a caddisfly, Hydropsyche sp. Using both our results and previously published literature
results, we characterize preservation effects across many different consumer taxa including invertebrates, fish, and
birds. Overall, only formalin fixation systematically affected isotope signature, causing an average depletion of 1.65‰
in δ13C, a bias that can easily be corrected for prior to interpreting data. Preservation affected mean δ15N values with
far lower frequency and magnitude, although variability increased with preservation for some taxa but not others. These
findings suggest that preserved specimens may be used for stable-isotope analysis and open up the possibility of using
archived collections to reconstruct food webs and biogeochemical changes at scales of tens to hundreds of years.
Résumé : L’analyse des isotopes stables est une méthode puissante qui permet de caractériser les flux de masse et
d’énergie dans les écosystèmes. Les études à long terme des réseaux alimentaires à l’aide d’isotopes stables sont précieuses, mais rares, car les échantillons requis ne sont pas facilement disponibles. Nous avons examiné la faisabilité
d’utiliser des spécimens conservés dans les collections d’histoire naturelle comme source de données à long terme pour
les analyses de réseaux alimentaires et nous avons vérifié si les agents de conservation chimique utilisés affectent les
signatures d’isotopes stables des tissus. Nous avons déterminé expérimentalement les effets de la conservation et de la
fixation dans l’éthanol 75 % et le formol 10 % sur *13C et *15N chez trois consommateurs aquatiques : le meunier de
Sacramento, Catostomus occidentalis, la moule asiatique, Corbicula fluminea, et le trichoptère, Hydropsyche sp. Nos
résultats et des travaux antérieurs dans la littérature nous ont permis de décrire les effets de la conservation chez un
grand nombre de taxons de consommateurs, dont des invertébrés, des poissons et des oiseaux. En général, seule la fixation au formol affecte systématiquement la signature isotopique, causant une réduction de 1,65 ‰ de *13C, une erreur
qui peut facilement être corrigée avant l’interprétation des données. La conservation affecte les valeurs moyennes de
*15N beaucoup moins fréquemment et moins fortement, bien que la variabilité augmente avec la durée de la conservation chez quelques taxons, mais pas chez d’autres. Ces résultats laissent espérer que des spécimens de collection pourront éventuellement être utilisés dans les analyses d’isotopes stables; on peut donc envisager la possibilité de se servir
des collections de musée pour reconstruire les réseaux alimentaires et les changements biogéochimiques sur des échelles de dizaines à des centaines d’années.
[Traduit par la Rédaction]
Sarakinos et al.
387
Received 25 May 2001. Accepted 10 January 2002. Published on the NRC Research Press Web site at http://cjz.nrc.ca on
8 March 2002.
H.C. Sarakinos1 and M.L. Johnson. John Muir Institute of the Environment, University of California, One Shields Avenue, Davis,
CA 95616, U.S.A.
M.J. Vander Zanden.2,3 Department of Environmental Science and Policy, University of California, One Shields Avenue, Davis,
CA 95616, U.S.A.
1
Present address: River Alliance of Wisconsin, 306 East Wilson, Suite 2W, Madison, WI 53703, U.S.A.
Corresponding author (e-mail: [email protected]).
3
Present address: Center for Limnology, 680 North Park Street, University of Wisconsin, Madison, WI 53706, U.S.A.
2
Can. J. Zool. 80: 381–387 (2002)
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Introduction
Stable-isotope analysis is a powerful method for characterizing flows of mass and energy through ecosystems because the stable-isotope composition of a consumer’s tissues
is related to that of its food (DeNiro and Epstein 1978,
1981; Minagawa and Wada 1984). Carbon-isotope ratios
(δ13C) change little from prey to predator, but vary among
primary producer groups and can thus acts as tracers of carbon source (Vander Zanden and Rasmussen 2001). Nitrogenisotope ratios (δ15N) exhibit consistent enrichment of 3–4‰
from prey to predator (Vander Zanden and Rasmussen
2001), providing a measure of a consumer’s trophic position
(Minagawa and Wada 1984; Cabana and Rasmussen 1994).
Typically, stable-isotope studies have provided qualitative
descriptions of trophic interactions in individual ecosystems.
Yet the importance of food-web studies at larger spatial and
temporal scales has been emphasized in the recent literature
(Pimm 1991; Polis and Winemiller 1996). Recent developments in baseline correction methods have facilitated comparative studies of food-web structure at broader spatial
scales (Cabana and Rasmussen 1996; Vander Zanden and
Rasmussen 1999). In contrast, studies of long-term trends
in food webs using stable-isotope analysis have rarely been
undertaken because of the lack of systematic, long-term
sampling at a given locale. Natural-history museums and
universities often house such site-specific collections in a
preserved state. If chemical preservation has no effect, or a
predictable effect, on stable-isotope composition of tissues,
retrospective analysis of archived samples will be a promising
tool for long-term studies of food-web and biogeochemical
processes at broader time scales of decades to centuries.
In this study we experimentally quantify effects of tissue
preservation on δ13C and δ15N signatures for three aquatic
consumer species. Using both our results and previously
published literature results, we characterize overall preservation effects and discuss some implications of the results for
long-term food-web studies.
Our experiments
We tested for effects of preservation in 70% ethanol (EtOH;
a preservative) and 4% formaldehyde (10% formalin; a
fixative). Effects were tested on isotope (δ13C and δ15N) signatures of three freshwater organisms: a fish, Catostomus
occidentalis, an aquatic insect, Hydropsyche sp., and a mollusk,
Corbicula fluminea. Organisms were collected in October
1999 and immediately processed in the laboratory. Dorsal
white-muscle tissue was removed from C. occidentalis.
Corbicula fluminea tissue was removed from the shell prior
to preservation. Hydropsyche sp. specimens were preserved
whole. All species underwent two preservation treatments
and a control treatment (–25°C storage). Treated tissues were
sampled at 3 days, 3 weeks, and 3 and 6 months. Fish and
invertebrate samples were dried at 75°C for 48–72 h and
pulverized using a mortar and pestle. The powder was packed
into 4 × 6 mm tin capsules for carbon- and nitrogen-isotope
analysis.
Stable-isotope analysis
Stable-isotope analysis was conducted on a continuousflow isotope-ratio mass spectrometer (dual-inlet Europa 20/20,
Can. J. Zool. Vol. 80, 2002
PDZ Europa, Crewe, England). Carbon and nitrogen stable
isotope ratios are expressed in delta (δ) notation, defined as
parts per thousand (‰, or per mil) deviations from a standard material. The standard is atmospheric nitrogen for δ15N
and Pee Dee belemnite (PDB) limestone for δ13C; both standards
have a δ value arbitrarily set at 0‰. Delta13C or δ15N =
[(Rsample/Rstandard) – 1 × 1000], where R = 13C/12C or 15N/14N.
A more positive “δ” value is isotopically enriched, meaning
that the sample contains proportionally more of the heavy
stable isotope (13C or 15N). Twenty-five percent of the samples were analyzed in duplicate; the mean error of our sample replicates was 0.09‰ for δ13C and 0.15‰ for δ15N.
Data analysis
We used paired t tests to test for the effect of EtOH and
formalin preservation on δ13C and δ15N of fish and invertebrate tissues. We used analysis of variance (ANOVA) to test
for effects of species and time and species-by-time interactions. Stated values represent the difference between control
and treatments; positive values indicate that preservation resulted in isotopic enrichment, while negative values indicate
isotopic depletion. Significance was based on Type III sums
of squares. Data were analyzed using SPSS for Windows
version 10.0 (SPSS Inc.). All stable-isotope values reported
below are calculated as the average over the 6-month duration of the experiment, i.e., the average of the four test dates.
Effects of EtOH and formalin
Only EtOH-preserved C. fluminea δ13C was enriched by
2.2‰ (p = 0.007) relative to control (Fig. 1a; Table 1). EtOH
preservation had a small though significant effect on δ15N of
C. occidentalis (0.37 ‰, p < 0.001) and C. fluminea (–0.39‰,
p = 0.03) (Fig. 1b; Table 1). Hydropsyche sp. δ15N was not
significantly affected (Fig. 1b; Table 1).
Formalin fixation affected the three species differently
(Fig. 1a). Overall, formalin-preserved Hydropsyche sp. and
C. occidentalis tissue δ13C was depleted relative to control
(–0.75 and –1.33‰, respectively, p = 0.00; Table 1). Corbicula
fluminea δ13C was enriched relative to control (0.67‰, p =
0.2), though this value was not statistically significant and
the slope of change fluctuated dramatically over time
(Fig. 1a; Table 1). Formalin affected samples rapidly (within
3 days to 3 weeks) except for Hydropsyche sp., where the effect appeared to increase with time (Fig. 1b). No significant
species-by-time interactions were observed. Formalin preservation had little effect on δ15N. Only C. fluminea tissue δ15N
was significantly depleted, by 0.48‰ (p = 0.002); δ15N of
Hydropsyche sp. and C. occidentalis did not differ from control (Fig. 1b; Table 1).
The broader context
Preservative effects on δ13C
Combining the results from our experiments and those
from previously published studies, we present the overall
impacts of EtOH and formalin on carbon-isotope ratios in
animal tissues (Table 1). Overall, EtOH had significant effects
on δ13C in 3 of 9 studies; only Drosophila melanogaster and
C. fluminea showed significant, though opposite, shifts in
δ13C relative to control. Drosophila melanogaster was depleted in δ13C by 1.3‰; C. fluminea was enriched in δ13C by
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Sarakinos et al.
383
Table 1. Effects of EtOH and formalin preservation on tissue δ13C and δ15N signatures.
EtOH
∆δ13Ca
∆δ15Na
Reference
0.27
0.08
0
–0.44
na
–0.10
–0.07
0.40
Gloutney and
Gloutney and
Gloutney and
Hobson et al.
–1.38*
–1.17*
0.21
2.18*
0.04
0.17
0.12
0.37*
–0.39*
–0.21
Ponsard and Amlou 1999
Ponsard and Amlou 1999
This study
This study
This study
18
–0.02 ± 0.24
17
0.04 ± 0.07
Quail
–0.16
–2.44*
–2.39*
–1.78*
n/a
0.03
0.24
0.04
Gloutney and
Gloutney and
Gloutney and
Hobson et al.
Fruit fly
–2.92*
–2.69*
–1.33*
0.67
–0.75*
–2.05
–0.74
–2.50c
na
0.34*
0.08
0.16
–0.48*
–0.121
0.35
1.21*
–1.00c
0.04
Ponsard and Amlou 1999
Ponsard and Amlou 1999
This study
This study
This study
Bosley and Wainright 1999
Bosley and Wainright 1999
Mullin et al. 1984
Toda and Wada 1990
21
–1.65 ± 0.24
22
0.08 ± 0.12
Scientific name
Common name
Cotrunix japonica
Egg, yolk lipidb
Egg, lipid-free yolkb
Egg, albuminb
C. japonicab
Drosophila melanogaster
10 daysb
6 weeksb
Catostomus occidentalis
Corbicula fluminea
Hydropsyche sp.
Quail
Fruit fly
Sacramento sucker
Asian clam
Caddisfly
n
Mean ± SE
Formalin
C. japonica
Egg, yolk lipidb
Egg, lipid-free yolkb
Egg, albuminb
C. japonicab
D. melanogaster
10 daysb
6 weeksb
C. occidentalis
C. fluminea
Hydropsyche sp.
Crangon septemspinosa
Pleuronectes americanus
Neomysis intermedia
Hobson 1998
Hobson 1998
Hobson 1998
1997
Sacramento sucker
Asian clam
Caddisfly
Mud shrimp
Winter flounder
Marine zooplankton
Freshwater shrimp
n
Mean ± SE
Hobson 1998
Hobson 1998
Hobson 1998
1997
a
Difference between control and treatment values. A positive value indicates that the treatment resulted in isotopic enrichment, a negative value denotes
the opposite.
b
No statistical information is available from this study.
c
Samples were rinsed in distilled water to remove preservative prior to desiccation.
*p < 0.05.
>2‰ (Table 1). Across all studies, formalin fixation resulted
in an average δ13C depletion of 1.65‰ (SE = 0.24‰), although some species were not significantly affected (Table 1). Additionally, organisms that were most depleted in
δ13C, Cotrunix japonica and its eggs and D. melanogaster,
were rinsed in distilled water prior to desiccation. This step
may have accelerated the breakdown of proteins, since bonds
that are formed between formaldehyde and proteins are unstable and easily dissociable (Taylor 1977; Stephenson and
Riley 1995).
EtOH and formalin may have affected preserved specimens
through two very different mechanisms. The first is selective
loss of material from the specimen during preservation. EtOH
hydrolyzes lipids, while formalin hydrolyzes protein (Von
Endt 1994; Hobson et al. 1997; Gloutney and Hobson 1998;
Bosley and Wainright 1999). Considering that lipids are depleted in δ13C relative to protein (DeNiro and Epstein 1977;
Von Endt 1994; Focken and Becker 1998), the observed depletion of δ13C in formalin-preserved specimens is consistent
with a loss of hydrolyzed proteins. However, differences in
lipid content among our three tested organisms did not explain any of the observed patterns in the effect of EtOH on
δ13C.
The second mechanism is uptake of the preservative into
the tissue. Both preservatives are carbon-based chemicals
with characteristic δ13C signatures. Once preserved samples
are immersed, their signature may shift toward that of the
preservative (Hobson et al. 1997; Gloutney and Hobson 1998;
Bosley and Wainright 1999; Ponsard and Amlou 1999). In
our experiment, δ13C in both fish and aquatic invertebrates
(–27 to –28‰) shifted toward that of the formalin medium
(–32‰) following fixation (Fig. 1b). No such shift was evident for EtOH.
Preservative effects on δ15N
Findings from our experiments and from the literature
(Mullin et al. 1984; Hobson et al. 1997; Gloutney and Hobson 1998; Ponsard and Amlou 1999) indicate a minimal effect of either preservative on δ15N across all species (mean
formalin effect: 0.10 ± 0.12‰; mean EtOH effect: 0.02 ±
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384
Can. J. Zool. Vol. 80, 2002
Fig. 1. Effect of EtOH (a) and formalin (b) on δ13C and δ15N of Hydropsyche sp., Corbicula fluminea, and Catostomus occidentalis
(䊉, control (frozen) values; 䊊, treatment values). Treatment length corresponds to the sequence in which treated samples were analyzed for isotope signature: 3 days, 3 weeks, 3 months, and 6 months. Results from two populations are shown for Hydropsyche sp.
(populations are seperated by the vertical line) and from three individuals for C. occidentalis (individuals are seperated by the vertical
lines). The dotted horizontal line in the δ13C plots corresponds to the signature of the preservative; there is no corresponding δ15N signature, since preservatives contain no nitrogen. Parentheses after treatment length in the x axis identify the individual populations (for
Hydropsyche) or specimens (for C. occidentalis).
(a)
Hydropsyche
8.5
-24
8.0
-26
-28
δ15 N
δ13 C
7.5
7.0
6.5
-30
6.0
-32
3d
k
3w
3m
o
6m
o
3d
(2
)
5.5
)
2)
2)
k (2
o( m o(
3w
3m
6
3d
k
3w
3m
o
6m
o
3d
(2
)
)
2)
2)
k (2
o( m o(
3w
3m
6
Treatment length
Treatment length
Corbicula fluminea
-24
8.5
8.0
-26
-28
δ15 N
δ13C
7.5
-30
7.0
6.5
6.0
-32
3d
3wk
3mo
5.5
6mo
3d
Treatment length
-24
3mo
6mo
Catostomus occidentalis
11.0
-26
10.5
-28
δ15 N
δ13C
3wk
Treatment length
10.0
-30
9.5
-32
3d 3wk 3mo 6mo d(2) k(2) o(2) o(2) d(3) k(3) o(3) o(3)
3 3w 3m 6m 3 3w 3m 6m
9.0
3d 3wk 3mo 6mo d(2) k(2) o(2) o(2) d(3) k(3) o(3) o(3)
3 3w 3m 6m 3 3w 3m 6m
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Sarakinos et al.
385
Fig. 1 (concluded).
Hydropsyche
(b)
8.5
-24
8.0
-26
-28
δ15 N
δ13 C
7.5
7.0
6.5
-30
6.0
-32
3d
k
3w
3m
o
6m
o
(
3d
2)
5.5
)
2)
2)
k (2
o( m o(
3w
3m
6
3d
k
3w
3m
o
6m
o
3d
(2
)
)
2)
2)
k (2
o( m o(
3w
3m
6
Treatment length
Treatment length
Corbicula fluminea
-24
8.5
8.0
-26
-28
δ15 N
δ13 C
7.5
-30
7.0
6.5
6.0
-32
3d
3wk
3mo
5.5
6mo
3d
Treatment length
-24
3mo
6mo
Catostomus occidentalis
11.0
-26
10.5
-28
15
δ N
δ13 C
3wk
Treatment length
10.0
-30
9.5
-32
3d 3wk 3mo 6mo d(2) k(2) o(2) o(2) d(3) k(3) o(3) o(3)
3 3w 3m 6m 3 3w 3m 6m
0.06‰; n = 23; Table 1). While some studies did show a
significant effect of preservative on δ15N, shifts in δ15N did
not exceed 0.5‰ except in formalin-preserved Pleuronectes
americanus, which was enriched by 1.4‰. Because both
EtOH and formalin contain no nitrogen, any change in δ15N
9.0
3d 3wk 3mo 6mo d(2) k(2) o(2) o(2) d(3) k(3) o(3) o(3)
3 3w 3m 6m 3 3w 3m 6m
following preservation would be due to tissue hydrolysis and
leaching, not to uptake of the N signature from the preservative. Bosley and Wainright (1999) observed that variability
in δ15N was greater in preserved Crangon septemspinosa
than in control C. septemspinosa (95% confidence intervals
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386
2.49 and 0.45, respectively). Similarly, we found that the
standard deviation of δ15N values for preserved C. occidentalis
samples was higher than that for controls (SDcontrol = 0.08‰,
SDEtOH = 0.165‰, SDformalin = 0.23‰).
Potential application to long-term ecological studies
Stable isotopes provide an integrative approach for measuring food-web interactions, although most food-web studies
have been conducted at relatively short time scales. Longer
term food-web studies can contribute valuable insights
(Wainright et al. 1993), but have rarely been conducted because the required samples are not available. Natural-history
museums house rich collections of animal specimens, often
collected over long time periods from a given location. As
our understanding of how isotope signatures are affected by
preservation builds, the potential exists to use long-preserved
specimens for stable-isotope analysis. Only formalin fixation
systematically affected isotope signature, producing a mean
depletion of 1.65‰ in δ13C, a bias that can easily be corrected for prior to data interpretation. The effects of EtOH
appear to be of lesser magnitude but are also less consistent.
While these results are promising, further research is needed
to verify that preservation effects remain similar at time
scales beyond those examined here (>6 months). These findings open up the possibility of reconstructing food-web and
biogeochemical changes over time scales of decades to centuries. This approach can be used to address a number of important questions, including the impacts of species invasion
(Vander Zanden et al. 1999) and movement of contaminants
through food webs (Cabana and Rasmussen 1994; Kidd et
al. 1995, 1998). Current approaches used for retrospective
food-web analyses are limited in their ability to resolve
changes at these time scales.
Global collections of formalin/EtOH-preserved specimens
currently stand at 1.5 billion and are growing at about 50
million a year (Peake 1989), constituting a significant portion of the record of the world’s documented biodiversity
(Von Endt 1994). Furthermore, many fields of biological research depend on the use of intact preserved specimens.
Practical applications such as those presented here augment
the value of natural-history collections and are a further incentive to continue the collection and maintenance of preserved archived samples of the world’s biodiversity.
Acknowledgements
We thank David Harris of the University of California
(UC) Davis Stable Isotope Facility for running the stableisotope analysis of the samples and Stephanie Carlson and
Sean Sollinger for assistance in sample collection and processing. We also thank Ron Cole, curator emeritus of the UC
Davis Wildlife and Fisheries Museum for access to spirit
collections literature. Funding was provided by the California
Department of Transportation through Contract No. 43A0014,
Task Order No. 4.
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